Particles for Inhalation Having Sustained Release Properties

ABSTRACT

The invention generally relates to a method for pulmonary delivery of therapeutic, prophylactic and diagnostic agents to a patient wherein the agent is released in a sustained fashion, and to particles suitable for use in the method. In particular, the invention relates to a method for the pulmonary delivery of a therapeutic, prophylactic or diagnostic agent comprising administering to the respiratory tract of a patient in need of treatment, prophylaxis or diagnosis an effective amount of particles comprising a therapeutic, prophylactic or diagnostic agent or any combination thereof in association with a charged lipid, wherein the charged lipid has an overall net charge which is opposite to that of the agent upon association with the agent. Release of the agent from the administered particles occurs in a sustained fashion.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/277,977, filed May 15, 2014, which is a continuation of U.S.application Ser. No. 13/220,334, filed Aug. 29, 2011, now abandoned,which is a continuation of U.S. application Ser. No. 11/523,914, filedSep. 20, 2006, now abandoned, which is a continuation of U.S.application Ser. No. 10/420,071, filed Apr. 18, 2003, now U.S. Pat. No.7,628,977, which is a continuation of application Ser. No. 09/752,106,filed Dec. 29, 2000, which is abandoned, which is a continuation-in-partof U.S. application Ser. No. 09/394,233, filed Sep. 13, 1999, now U.S.Pat. No. 6,652,837, which is a continuation-in-part of application Ser.No. 08/971,791, filed Nov. 17, 1997, now U.S. Pat. No. 5,985,309, whichclaims the benefit of U.S. Provisional Application No. 60/059,004, filedSep. 15, 1997 and which is a continuation of U.S. application Ser. No.08/784,421 filed Jan. 16, 1997, issued as U.S. Pat. No. 5,855,913 andreissued as U.S. RE 37,053, which is a continuation-in-part of U.S.application Ser. No. 08/739,308, filed on Oct. 29, 1996, now U.S. Pat.No. 5,847,064, which is a continuation-in-part of U.S. application Ser.No. 08/655,570 filed on May 24, 1996, which is abandoned.

This application is also related to application Ser. No. 09/337,245,filed Jun. 22, 1999; Ser. No. 09/383,054, filed on Aug. 25, 1999; Ser.No. 09/382,959, filed Aug. 25, 1999; Ser. No. 09/644,320, filed on Aug.23, 2000; Ser. No. 09/665,252, filed Sep. 19, 2000, now U.S. Pat. No.6,514,482; Ser. No. 09/644,105, filed Aug. 23, 2000; Ser. No.09/644,736, filed Aug. 23, 2000; and Ser. No. 09/591,307, filed Jun. 9,2000. The entire teachings of the above applications are incorporatedherein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No.HD029129, awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

Pulmonary delivery of bioactive agents, for example, therapeutic,diagnostic and and prophylactic agents provides an attractivealternative to, for example, oral, transdermal and parenteraladministration. That is, pulmonary administration can typically becompleted without the need for medical intervention(self-administration), the pain often associated with injection therapyis avoided, and the amount of enzymatic and pH mediated degradation ofthe bioactive agent, frequently encountered with oral therapies, can besignificantly reduced. In addition, the lungs provide a large mucosalsurface for drug absorption and there is no first-pass liver effect ofabsorbed drugs. Further, it has been shown that high bioavailability ofmany molecules, for example, macromolecules, can be achieved viapulmonary delivery or inhalation. Typically, the deep lung, or alveoli,is the primary target of inhaled bioactive agents, particularly foragents requiring systemic delivery.

The release kinetics or release profile of a bioactive agent into thelocal and/or systemic circulation is a key consideration in mosttherapies, including those employing pulmonary delivery. That is, manyillnesses or conditions require administration of a constant orsustained levels of a bioactive agent to provide an effective therapy.Typically, this can be accomplished through a multiple dosing regimen orby employing a system that releases the medicament in a sustainedfashion.

However, delivery of bioactive agents to the pulmonary system typicallyresults in rapid release of the agent following administration. Forexample, U.S. Pat. No. 5,997,848 to Patton et al. describes the rapidabsorption of insulin following administration of a dry powderformulation via pulmonary delivery. The peak insulin level was reachedin about 30 minutes for primates and in about 20 minutes for humansubjects. Further, Heinemann, Traut and Heise teach in Diabetic Medicine14:63-72 (1997) that the onset of action, assessed by glucose infusionrate, in healthy volunteers after inhalation was rapid with thehalf-maximal action reached in about 30 minutes.

As such, a need exists for formulations suitable for inhalationcomprising bioactive agents and wherein the bioactive agent of theformulation is released in a sustained fashion into the systemic and/orlocal circulation.

SUMMARY OF THE INVENTION

This invention is based upon the unexpected discovery that combining acharged agent with a lipid carrying an opposite charge results in asustained release profile of the agent.

The invention generally relates to a method for pulmonary delivery oftherapeutic, prophylactic and diagnostic agents to a patient wherein theagent is released in a sustained fashion, and to particles suitable foruse in the method. In particular, the invention relates to a method forthe pulmonary delivery of a therapeutic, prophylactic or diagnosticagent comprising administering to the respiratory tract of a patient inneed of treatment, prophylaxis or diagnosis an effective amount ofparticles comprising a therapeutic, prophylactic or diagnostic agent orany combination thereof in association with a charged lipid, wherein thecharged lipid has an overall net charge which is opposite to that of theagent upon association with the agent. Release of the agent from theadministered particles occurs in a sustained fashion.

In one embodiment, the association of the therapeutic, prophylactic ordiagnostic agent and the oppositely charged lipid can result from ioniccomplexation. In another embodiment, association of the therapeutic,prophylactic or diagnostic agent and the oppositely charged lipid canresult from hydrogen bonding.

In yet a further embodiment, the association of the therapeutic,prophylactic or diagnostic agent and the oppositely charged lipid canresult from a combination of ionic complexation and hydrogen bonding.

The particles suitable for use in the method can comprise a therapeutic,prophylactic or diagnostic agent in association with a charged lipidhaving a charge opposite to that of the agent. The charges are oppositeupon association, prior to administration. In a preferred embodiment,the charges of the agent and lipid upon association, prior toadministration, are those which the agent and lipid possess at pulmonarypH.

For example, the particles suitable for pulmonary delivery can comprisea therapeutic, prophylactic or diagnostic agent which possesses anoverall net negative charge, in association with a lipid which possessesan overall net positive charge. For example, the agent can be insulinwhich has an overall net charge which is negative and the lipid can be1,2-dipalmitoyl-sn-glycero-3-ethylphosphatidylcholine (DPePC).

Alternatively, the particles suitable for pulmonary delivery cancomprise a therapeutic, prophylactic or diagnostic agent which possessesan overall net positive charge in association with a lipid whichpossesses an overall net negative charge. For example, the agent can bealbuterol which possesses an overall positive charge and the lipid canbe 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)](DPPG) whichpossesses an overall net negative charge.

Further, the particles suitable for pulmonary delivery can comprise atherapeutic, prophylactic or diagnostic agent which has an overall netcharge which can be modified by adjusting the pH of a solution of theagent, prior to association with the lipid. For example, at a pH ofabout 7.4, insulin has an overall net charge which is negative.Therefore, insulin and a positively charged lipid can be associated atthis pH prior to administration to prepare a particle having an agent inassociation with a charged lipid wherein the charged lipid has a chargeopposite to that of the agent. However, the charges on insulin can alsobe modified, when in solution, to possess an overall net charge which ispositive by modifying the pH of the solution to be less than the pI ofinsulin (pI=5.5). As such, when insulin is in solution at a pH of about4, for example, it will possess an overall net charge which is positive.As this is the case, the positively charged insulin can be associatedwith a negatively charged lipid, for example,1,2-distearoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DSPG).

Modification of the charge of the therapeutic, prophylactic ordiagnostic agent prior to association with the charged lipid, can beaccomplished with many agents, particularly, proteins. For example,charges on proteins can be modulated by spray drying feed solutionsbelow or above the isoelectric points (pI) of the protein. Chargemodulation can also be accomplished for small molecules by spray dryingfeed solutions below or above the pKa of the molecule.

In a particular embodiment, the particles of the invention comprise morethan one lipid, more than one bioactive agent or both. Also chargedlipids can be combined with lipids without a net charge.

The particles, can further comprise a carboxylic acid which is distinctfrom the bioactive agent and lipid. In one embodiment, the carboxylicacid includes at least two carboxyl groups. Carboxylic acids, includethe salts thereof as well as combinations of two or more carboxylicacids and/or salts thereof. In a preferred embodiment, the carboxylicacid is a hydrophilic carboxylic acid or salt thereof. Citric acid andcitrates, such as, for example sodium citrate, are preferred.Combinations or mixtures of carboxylic acids and/or their salts also canbe employed.

The particles suitable for use in the invention can further comprise amultivalent salt or its ionic components. In a preferred embodiment, thesalt is a divalent salt. In another preferred embodiment, the salt is asalt of an alkaline-earth metal, such as, for example, calcium chloride.The particles of the invention can also include mixtures or combinationsof salts and/or their ionic components.

The particles suitable for use in the invention can further comprise anamino acid. In a preferred embodiment the amino acid is hydrophobic.

The particles, also referred to herein as powder, can be in the form ofa dry powder suitable for inhalation. The particles can have a tapdensity of less than about 0.4 g/cm³, preferably less than about 0.1g/cm³. Further, the particles suitable for use in the invention can havea median geometric diameter of from about 5 micrometers to about 30micrometers. In yet another embodiment, the particles suitable for usein the invention have an aerodynamic diameter of from about 1 to about 5micrometers.

The invention has numerous advantages. For example, particles suitablefor inhalation can be designed to possess a sustained release profile.This sustained released profile provides for prolonged residence of theadministered bioactive agent in the lung and increases the amount oftime in which therapeutic levels of the agent are present in the localenvironment or systemic circulation. The sustained release of agentprovides a desirable alternative to injection therapy currently used formany therapeutic, diagnostic and prophylactic agent requiring sustainedrelease of agent, such as insulin for the treatment of diabetes. Inaddition, the invention provides a method of delivery to the pulmonarysystem wherein the high initial release of agent typically seen ininhalation therapy is reduced. Consequently, patient compliance andcomfort can be increased by not only reducing frequency of dosing, butby providing a therapy which is more amenable to patients.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1 is a graph showing the in vivo release profile of dry powderformulations comprising insulin and either a lipid (DPPC) with nooverall net a charge or lipid having an overall net charge opposite tothat of insulin (DSePC and DPePC).

FIG. 2 is a graph of the in vivo release profile of dry powderformulations comprising insulin in combination with a lipid with nooverall net charge (DPPC) or insulin in combination with a charged lipidhaving an overall negative charge (DPPG) and spray dried with the activeagent at either pH 4 or 7.4.

FIG. 3 is a graph showing the in vivo release profile of dry powderformulation comprising estrone sulfate (−) and either a lipid with nooverall net charge (DPPC) or a lipid having an overall charge opposite(DPePC, +) to that of the estrone sulfate.

FIG. 4 is a graph of percent of PenH above baseline versus timefollowing administration of dry powder formulations of albuterol sulfateand lipid in animals which have been challenged repeatedly over timewith methacholine given by nebulization.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

Therapeutic, prophylactic or diagnostic agents, can also be referred toherein as “bioactive agents,” “medicaments” or “drugs.”

The invention relates to a method for the pulmonary delivery oftherapeutic, prophylactic and diagnostic agents comprising administeringto the respiratory tract of a patient in need of treatment, prophylaxisor diagnosis an effective amount of particles comprising a therapeutic,prophylactic or diagnostic agent or any combination thereof inassociation with a charged lipid, wherein the charged lipid has anoverall net charge which is opposite to that of the agent. The agent isreleased from the administered particles in a sustained fashion.

The particles of the invention release bioactive agent in a sustainedfashion. As such, the particles possess sustained release properties.“Sustained release”, as that term is used herein, refers to a release ofactive agent in which the period of release of an effective level ofagent is longer than that seen with the same bioactive agent which isnot associated with an oppositely charged lipid, prior toadministration. In addition, a sustained release also refers to areduction in the burst of agent typically seen in first two hoursfollowing administration, and more preferably in the first hour, oftenreferred to as the initial burst. In a preferred embodiment, thesustained release is characterized by both the period of release beinglonger in addition to a decreased burst. For example, a sustainedrelease of insulin can be a release showing elevated levels out to atleast 4 hours post administration, such as about 6 hours or more.

“Pulmonary delivery,” as that term is used herein refers to delivery tothe respiratory tract. The “respiratory tract,” as defined herein,encompasses the upper airways, including the oropharynx and larynx,followed by the lower airways, which include the trachea followed bybifurcations into the bronchi and bronchioli (e.g., terminal andrespiratory). The upper and lower airways are called the conductingairways. The terminal bronchioli then divide into respiratory bronchioliwhich then lead to the ultimate respiratory zone, namely, the alveoli,or deep lung. The deep lung, or alveoli, are typically the desired thetarget of inhaled therapeutic formulations for systemic drug delivery.

In one embodiment, the therapeutic, prophylactic or diagnostic agent andthe oppositely charged lipid can be in association primarily as a resultof ionic bonding, for example, ionic complexation. In anotherembodiment, the therapeutic, prophylactic or diagnostic agent and theoppositely charged lipid can be in association primarily as a result ofhydrogen bonding. It is understood that a combination of ionic andhydrogen bonding can contribute to the association of the bioactive andcharged lipid.

Ionic bonding is bonding which occurs via charge/charge interactionsbetween atoms or groups of atoms. Since opposite charges attract, theatoms in an ionic compound are held together by this attraction.

Hydrogen bonding refers to bonding wherein a hydrogen atom is sharedbetween two molecules. For example, a hydrogen atom covalently attachedto an electronegative atom such as nitrogen, oxygen, sulfur orphosphorous shares its partial positive charge with a secondelectronegative atom, for example, nitrogen, oxygen, sulfur orphosphorous.

The particles suitable for use in the method can comprise a therapeutic,prophylactic or diagnostic agent in association with a charged lipidhaving a charge opposite to that of the agent upon association, prior toadministration. In a preferred embodiment, the charges possessed by theagent and lipid, upon association, are the same as the charges which theagent and lipid possess at pulmonary pH following administration.

For example, the particles suitable for pulmonary delivery can comprisea therapeutic, prophylactic or diagnostic agent which possesses anoverall net negative charge in association with a lipid which possessesan overall net positive charge. For example, the agent can be insulinand the lipid can be an alkylphosphatidylcholine, such as1,2-dipalmitoyl-sn-glycero-3-ethylphosphatidylcholine (DPePC).

Alternatively, the particles suitable for pulmonary delivery cancomprise a therapeutic, prophylactic or diagnostic agent which possessesan overall net positive charge in association with a lipid whichpossesses an overall net negative charge, preferably in the pulmonary pHrange. For example, the agent can be albuterol sulfate which possessesan overall positive charge and the lipid can be1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) whichpossesses an overall net negative charge.

Further, the particles suitable for pulmonary delivery can comprise atherapeutic, prophylactic or diagnostic agent which has an overall netcharge which can be modified by adjusting the pH of a solution of theagent prior to association with the charged lipid. For example, at a pHof about 7.4 insulin has an overall net charge which is negative.Therefore, insulin and a positively charged lipid can be associated atthis pH, prior to administration, to prepare a particle having abioactive agent in association with a charged lipid wherein the chargedlipid has a charge opposite to that of the agent upon association.However, insulin can also be modified when in solution to possess anoverall net charge which is positive by modifying the pH of the solutionto be less than the pI of insulin (pI=5.5). As such, when insulin is insolution at a pH of 4, for example, it will possess an overall netcharge which is positive. As this is the case, the positively chargedinsulin can be associated with a negatively charged lipid, for example,1,2-distearoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DSPG).Modification of the charge of the therapeutic, prophylactic ordiagnostic agent is applicable to many agents, particularly, proteins.

“Pulmonary pH range”, as that term is used herein, refers to the pHrange which can be encountered in the lung of a patient. Typically, inhumans, this range of pH is from about 6.4 to about 7.0, such as from6.4 to about 6.7. pH values of the airway lining fluid (ALF) have beenreported in “Comparative Biology of the Normal Lung”, CRC Press, (1991)by R. A. Parent and range from 6.44 to 6.74.

“Charged lipid” as that term is used herein, refers to lipids which arecapable of possessing an overall net charge. The charge on the lipid canbe negative or positive. The lipid can be chosen to have a chargeopposite to that of the active agent when the lipid and active agent areassociated. In a preferred embodiment the charged lipid is a chargedphospholipid. Preferably, the phospholipid is endogenous to the lung orcan be metabolized upon administration to a lung endogenousphospholipid. Combinations of charged lipids can be used. Thecombination of charged lipid also has an overall net charge opposite tothat of the bioactive agent upon association.

The charged phospholipid can be a negatively charged lipid such as, a1,2-diacyl-sn-glycero-3-[phospho-rac-(1-glycerol)] and a1,2-diacyl-sn-glycerol-3-phosphate.

The 1,2-diacyl-sn-glycero-3-[phospho-rac-(1-glycerol)] phospholipids canbe represented by the Formula I:

wherein R₁ and R₂ are independently aliphatic groups having from about 3to about 24 carbon atoms, preferably from about 10 to about 20 carbonatoms.

Aliphatic group as that term is used herein in Formulas I-VI refers tosubstituted or unsubstituted straight chained, branched or cyclic C₁-C₂₄hydrocarbons which can be completely saturated, which can contain one ormore heteroatoms such as nitrogen, oxygen or sulfur and/or which cancontain one or more units of unsaturation.

Suitable substituents on an aliphatic group include —OH, halogen (—Br,—Cl, —I and —F) —O (aliphatic, substituted), —CN, —NO₂, —COOH, —NH₂, —NH(aliphatic group, substituted aliphatic), —N(aliphatic group,substituted aliphatic group)₂, —COO (aliphatic group, substitutedaliphatic group), —CONH₂, —CONH (aliphatic, substituted aliphaticgroup), —SH, —S(aliphatic, substituted aliphatic group) and—NH—C(═NH)—NH₂. A substituted aliphatic group can also have a benzyl,substituted benzyl, aryl (e.g., phenyl, naphthyl or pyridyl) orsubstituted aryl group as a substituent. A substituted aliphatic canhave one or more substituents.

Specific examples of this type of negatively charged phospholipidinclude, but are not limited to,1,2-distearoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DSPG),1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG),1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol)] (DPPG),1,2-dilauroyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DLPG), and1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG).

The 1,2-diacyl-sn-glycerol-3-phosphate phospholipids can be representedby the Formula II:

R₁ and R₂ are independently an aliphatic group having from about 3 toabout 24 carbon atoms, preferably from about 10 to about 20 carbonatoms.

Specific examples of this type of phospholipid include, but are notlimited to, 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA),1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA),1,2-dioleoyl-sn-glycero-3-phosphate (DOPA),1,2-distearoyl-sn-glycero-3-phosphate (DSPA), and1,2-dilauroyl-sn-glycero-3-phosphate (DLPA).

The charged lipid can be a positively charged lipid such as a1,2-diacyl-sn-glycero-3-alkylphosphocholine and a1,2-diacyl-sn-glycero-3-alkylphosphoalkanolamine.

The 1,2-diacyl-sn-glycero-3-alkyllphosphocholine phospholipids can berepresented by the Formula III:

wherein R₁ and R₂ are independently an aliphatic group having from about3 to about 24 carbon atoms, preferably from about 10 to about 20 carbonatoms. R₃ is an aliphatic group having from about 1 to about 24 carbons,for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyland the like.

Specific examples of this type of positively charged phospholipidinclude, but are not limited to,1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPePC),1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMePC),1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSePC),1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLePC), and1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOePC). The1,2-diacyl-sn-glycero-3-alkylphosphoalkanolamine phospholipids can berepresented by the Formula IV:

wherein R₁ and R₂ are independently an aliphatic group having from about3 to about 24 carbon atoms, preferably from about 10 to about 20 carbonatoms. R₃ is an aliphatic group having from about 1 to about 24 carbons,for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyland the like. R₄ is independently hydrogen, or an aliphatic group havingfrom about 1 to about 6 carbon atoms.

Specific examples of this type of positively charged phospholipidinclude, but are not limited to,1,2-dipalmitoyl-sn-glycero-3-ethylethanolamine (DPePE),1,2-dimyristoyl-sn-glycero-3-ethylphosphoethanolamine (DMePE),1,2-distearoyl-sn-glycero-3-ethylphosphoethanolamine (DSePE),1,2-dilauroyl-sn-glycero-3-ethylphosphoethanolamine (DLePE), and1,2-dioleoyl-sn-glycero-3-ethylphosphoethanolamine (DOePE).

Other charged lipids suitable for use in the invention include thosedescribed in U.S. Pat. No. 5,466,841 to Horrobin et al. issued on Nov.14, 1995, U.S. Pat. Nos. 5,698,721 and 5,902,802 to Heath issued Dec.16, 1997 and May 11, 1999, respectively, and U.S. Pat. No. 4,480,041 toMyles et al. issued Oct. 30, 1984, the entire contents of all of whichare incorporated herein by reference.

The charged lipid and the therapeutic, prophylactic or diagnostic agentcan be present in the particles of the invention at a charge ratio oflipid to active of from about 0.25:1 or more, preferably from about0.25:1 to about 1:0.25, for example, about 0.5:1 to about 1:0.5.Preferably the charge ratio is about 1:1. When an excess of charge ispresent, it is preferred that the excess charge is contributed by thelipid.

A suitable charge ratio can be determined as follows. First, the numberof charges present on both the bioactive agent and lipid, at theconditions under which association of the two will occur, prior toadministration, should be determined. Next, the equivalent weight ofboth the bioactive agent and lipid should be determined. This can becarried out following the example below employing insulin as thebioactive agent and DPePC as the charged lipid at a pH of about 7.4.

-   -   Molecular Weight of Insulin: 5,800 g/mole    -   Number of Negative Charges on Insulin: 6 equivalent    -   Equivalent Weight Per Charge: 5,800×1/6=967 g    -   Molecular Weight of DPePC: 763 g/mole    -   Number of Negative Charges on DPePC: 1 equivalent    -   Equivalent Weight Per Charge: 763×1/1=763 g        Therefore, to obtain for example, a 1:1 charge ratio of DPePC to        insulin    -   763 g DPePC is associated with 967 g insulin        -   OR    -   1 g DPePC is associated with 1.27 (967/763=1.27) g insulin.

Alternatively,

-   -   967 g insulin is associated with 763 g DPePC        -   OR    -   1 g insulin is associated with 0.79 (763/967=0.79) g DPePC.        In molar terms,    -   1 mole DPePC is associated with 1/6 mole insulin        -   OR    -   1 mole insulin is associated with 6 moles DPePC.

This analysis can be used to determine the amount of lipid and activeagent needed for any ratio desired and any combination of bioactiveagent and lipid.

The charged lipid can be present in the particles in an amount rangingfrom about 1 to about 99% by weight. Preferably, the charged lipid ispresent in the particles in an amount ranging from about 10% to about90% by weight.

The particles of the invention can also comprise phospholipids, whichare zwitterionic and therefore do not possess an overall net charge.Such lipids, can assist in providing particles with the propercharacterisitics for inhalation. Such phospholipids suitable for use inthe invention include, but are not limited to, a1,2-diacyl-sn-glycero-3-phosphocholine and a1,2-diacyl-sn-glycero-3-phosphoalkanolamine. These lipids can preferablybe present in the particles in an amount ranging from about 10% to about90% by weight. Preferably, these lipids can be present in the particlesin an amount ranging from abut 50% to about 80% by weight.

The 1,2-diacyl-sn-glycero-3-phosphocholine phospholipids can berepresented by Formula V:

R₁ and R₂ are independently an aliphatic group having from about 3 toabout 24 carbon atoms, preferably from about 10 to about 20 carbonatoms. R₄ is independently hydrogen, or an aliphatic group having fromabout 1 to about 6 carbon atoms.

Specific examples of 1,2-diacyl-sn-glycero-3-phosphocholinephospholipids include, but are not limited to,1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dilaureoyl-sn-3-glycero-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),

The 1,2-diacyl-sn-glycero-3-phosphoalkanolamine phospholipids can berepresented by Formula VI:

wherein R₁ and R₂ are independently an aliphatic group having from about3 to about 24 carbon atoms, preferably, from about 10 to about 20 carbonatoms and R₄ is independently hydrogen or an aliphatic group having fromabout 1 to about 6 carbon atoms.

Specific examples of this type of phospholipid include, but are notlimited to, 1,2-dipalmitoyl-sn-glycero-3-ethanolamine (DPPE),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), and1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

Therapeutic, prophylactic or diagnostic agents, can also be referred toherein as “bioactive agents,” “medicaments” or “drugs.” It is understoodthat one or more bioactive agents can be present in the particles of theinvention. Hydrophilic as well as hydrophobic agents can be used. Theagent must be capable of possessing an overall net charge. The amount ofbioactive agent present in the particles of the invention can be fromabout 0.1 weight % to about 95 weight %, for example, from about 5 toabout 75%, such as from about 10 to about 50%. Particles in which thedrug is distributed throughout a particle are preferred.

Suitable bioactive agents include agents which can act locally,systemically or a combination thereof. The term “bioactive agent,” asused herein, is an agent, or its pharmaceutically acceptable salt, whichwhen released in vivo, possesses the desired biological activity, forexample therapeutic, diagnostic and/or prophylactic properties in vivo.

Examples of bioactive agent include, but are not limited to, syntheticinorganic and organic compounds, proteins and peptides, polysaccharidesand other sugars, lipids, and DNA and RNA nucleic acid sequences havingtherapeutic, prophylactic or diagnostic activities. Agents with a widerange of molecular weight can be used, for example, between 100 and500,000 grams or more per mole.

The agents can have a variety of biological activities, such asvasoactive agents, neuroactive agents, hormones, anticoagulants,immunomodulating agents, cytotoxic agents, prophylactic agents,antibiotics, antivirals, antisense, antigens, antineoplastic agents andantibodies.

Proteins, include complete proteins, muteins and active fragmentsthereof, such as insulin, immunoglobulins, antibodies, cytokines (e.g.,lymphokines, monokines, chemokines), interleukins, interferons (β-IFN,α-IFN and γ-IFN), erythropoietin, nucleases, tumor necrosis factor,colony stimulating factors, enzymes (e.g. superoxide dismutase, tissueplasminogen activator), tumor suppressors, blood proteins, hormones andhormone analogs (e.g., growth hormone, adrenocorticotropic hormone andluteinizing hormone releasing hormone (LHRH)), vaccines (e.g., tumoral,bacterial and viral antigens), antigens, blood coagulation factors;growth factors; granulocyte colony-stimulating factor (“G-CSF”);peptides include protein inhibitors, protein antagonists, and proteinagonists, calcitonin; nucleic acids include, for example, antisensemolecules, oligonucleotides, and ribozymes. Polysaccharides, such asheparin, can also be administered.

Bioactive agent for local delivery within the lung, include such asagents as those for the treatment of asthma, chronic obstructivepulmonary disease (COPD), emphysema, or cystic fibrosis. For example,genes for the treatment of diseases such as cystic fibrosis can beadministered, as can beta agonists steroids, anticholinergics, andleukotriene modifers for asthma.

Other specific bioactive agents include, estrone sulfate, albuterolsulfate, parathyroid hormone-related peptide, somatostatin, nicotine,clonidine, salicylate, cromolyn sodium, salmeterol, formeterol, L-dopa,Carbidopa or a combination thereof, gabapenatin, clorazepate,carbamazepine and diazepam.

Nucleic acid sequences include genes, antisense molecules which can, forinstance, bind to complementary DNA to inhibit transcription, andribozymes.

The particles can include any of a variety of diagnostic agents tolocally or systemically deliver the agents following administration to apatient. For example, imaging agents which include commerciallyavailable agents used in positron emission tomography (PET), computerassisted tomography (CAT), single photon emission computerizedtomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI) canbe employed.

Examples of suitable materials for use as contrast agents in MRI includethe gadolinium chelates currently available, such as diethylene triaminepentacetic acid (DTPA) and gadopentotate dimeglumine, as well as iron,magnesium, manganese, copper and chromium.

Examples of materials useful for CAT and x-rays include iodine basedmaterials for intravenous administration, such as ionic monomerstypified by diatrizoate and iothalamate and ionic dimers, for example,ioxagalte.

Diagnostic agents can be detected using standard techniques available inthe art and commercially available equipment.

The particles can further comprise a carboxylic acid which is distinctfrom the agent and lipid. In one embodiment, the carboxylic acidincludes at least two carboxyl groups. Carboxylic acids, include thesalts thereof as well as combinations of two or more carboxylic acidsand/or salts thereof. In a preferred embodiment, the carboxylic acid isa hydrophilic carboxylic acid or salt thereof. Suitable carboxylic acidsinclude but are not limited to hydroxydicarboxylic acids,hydroxytricarboxilic acids and the like. Citric acid and citrates, suchas, for example sodium citrate, are preferred. Combinations or mixturesof carboxylic acids and/or their salts also can be employed.

The carboxylic acid can be present in the particles in an amount rangingfrom about 0 to about 80% weight. Preferably, the carboxylic acid can bepresent in the particles in an amount of about 10 to about 20%.

The particles suitable for use in the invention can further comprise amultivalent salt or its ionic components. As used herein, a“multivalent” salt refers to salts having a ionic component with avalency greater than one. For example, divalent salts. In a preferredembodiment, the salt is a divalent salt. In another preferredembodiment, the salt is a salt of an alkaline-earth metal, such as, forexample, calcium chloride. The particles of the invention can alsoinclude mixtures or combinations of salts and/or their ionic components.

The salt or its ionic components are present in the particles in anamount ranging from about 0 to about 40% weight.

The particles suitable for use in the invention can further comprise anamino acid. In a preferred embodiment the amino acid is hydrophobic.Suitable naturally occurring hydrophobic amino acids, include but arenot limited to, leucine, isoleucine, alanine, valine, phenylalanine,glycine and tryptophan. Combinations of hydrophobic amino acids can alsobe employed. Non-naturally occurring amino acids include, for example,beta-amino acids. Both D, L configurations and racemic mixtures ofhydrophobic amino acids can be employed. Suitable hydrophobic aminoacids can also include amino acid derivatives or analogs. As usedherein, an amino acid analog includes the D or L configuration of anamino acid having the following formula: —NH—CHR—CO—, wherein R is analiphatic group, a substituted aliphatic group, a benzyl group, asubstituted benzyl group, an aromatic group or a substituted aromaticgroup and wherein R does not correspond to the side chain of anaturally-occurring amino acid. As used herein, aliphatic groups includestraight chained, branched or cyclic C1-C8 hydrocarbons which arecompletely saturated, which contain one or two heteroatoms such asnitrogen, oxygen or sulfur and/or which contain one or more units ofunsaturation. Aromatic or aryl groups include carbocyclic aromaticgroups such as phenyl and naphthyl and heterocyclic aromatic groups suchas imidazolyl, indolyl, thienyl, furanyl, pyridyl, pyranyl, oxazolyl,benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl and acridintyl.

Suitable substituents on an aliphatic, aromatic or benzyl group include—OH, halogen (—Br, —Cl, —I and —F) —O(aliphatic, substituted aliphatic,benzyl, substituted benzyl, aryl or substituted aryl group), —CN, —NO₂,—COOH, —NH₂, —NH (aliphatic group, substituted aliphatic, benzyl,substituted benzyl, aryl or substituted aryl group), —N(aliphatic group,substituted aliphatic, benzyl, substituted benzyl, aryl or substitutedaryl group)₂, —COO (aliphatic group, substituted aliphatic, benzyl,substituted benzyl, aryl or substituted aryl group), —CONH₂, —CONH(aliphatic, substituted aliphatic group, benzyl, substituted benzyl,aryl or substituted aryl group)), —SH, —S(aliphatic, substitutedaliphatic, benzyl, substituted benzyl, aromatic or substituted aromaticgroup) and —NH—C(═NH)—NH₂. A substituted benzylic or aromatic group canalso have an aliphatic or substituted aliphatic group as a substituent.A substituted aliphatic group can also have a benzyl, substitutedbenzyl, aryl or substituted aryl group as a substituent. A substitutedaliphatic, substituted aromatic or substituted benzyl group can have oneor more substituents. Modifying an amino acid substituent can increase,for example, the lypophilicity or hydrophobicity of natural amino acidswhich are hydrophilic.

A number of the suitable amino acids, amino acids analogs and saltsthereof can be obtained commercially. Others can be synthesized bymethods known in the art. Synthetic techniques are described, forexample, in Green and Wuts, “Protecting Groups in Organic Synthesis”,John Wiley and Sons, Chapters 5 and 7, 1991.

Hydrophobicity is generally defined with respect to the partition of anamino acid between a nonpolar solvent and water. Hydrophobic amino acidsare those acids which show a preference for the nonpolar solvent.Relative hydrophobicity of amino acids can be expressed on ahydrophobicity scale on which glycine has the value 0.5. On such ascale, amino acids which have a preference for water have values below0.5 and those that have a preference for nonpolar solvents have a valueabove 0.5. As used herein, the term hydrophobic amino acid refers to anamino acid that, on the hydrophobicity scale has a value greater orequal to 0.5, in other words, has a tendency to partition in thenonpolar acid which is at least equal to that of glycine.

Examples of amino acids which can be employed include, but are notlimited to: glycine, proline, alanine, cysteine, methionine, valine,leucine, tyrosine, isoleucine, phenylalanine, tryptophan. Preferredhydrophobic amino acids include leucine, isoleucine, alanine, valine,phenylalanine, glycine and tryptophan. Combinations of hydrophobic aminoacids can also be employed. Furthermore, combinations of hydrophobic andhydrophilic (preferentially partitioning in water) amino acids, wherethe overall combination is hydrophobic, can also be employed.Combinations of one or more amino acids can also be employed.

The amino acid can be present in the particles of the invention in anamount from about 0% to about 60 weight %. Preferably, the amino acidcan be present in the particles in an amount ranging from about 5 toabout 30 weight %. The salt of a hydrophobic amino acid can be presentin the particles of the invention in an amount of from about 0% to about60 weight %. Preferably, the amino acid salt is present in the particlesin an amount ranging from about 5 to about 30 weight %. Methods offorming and delivering particles which include an amino acid aredescribed in U.S. patent application Ser. No. 09/382,959, filed on Aug.25, 1999, entitled Use of Simple Amino Acids to Form Porous ParticlesDuring Spray Drying the entire teaching of which is incorporated hereinby reference.

In a further embodiment, the particles can also include other materialssuch as, for example, buffer salts, dextran, polysaccharides, lactose,trehalose, cyclodextrins, proteins, peptides, polypeptides, fatty acids,fatty acid esters, inorganic compounds, phosphates.

In one embodiment of the invention, the particles can further comprisepolymers. The use of polymers can further prolong release. Biocompatibleor biodegradable polymers are preferred. Such polymers are described,for example, in U.S. Pat. No. 5,874,064, issued on Feb. 23, 1999 toEdwards et al., the teachings of which are incorporated herein byreference in their entirety.

In yet another embodiment, the particles include a surfactant other thanone of the charged lipids described above. As used herein, the term“surfactant” refers to any agent which preferentially absorbs to aninterface between two immiscible phases, such as the interface betweenwater and an organic polymer solution, a water/air interface or organicsolvent/air interface. Surfactants generally possess a hydrophilicmoiety and a lipophilic moiety, such that, upon absorbing tomicroparticles, they tend to present moieties to the externalenvironment that do not attract similarly-coated particles, thusreducing particle agglomeration. Surfactants may also promote absorptionof a therapeutic or diagnostic agent and increase bioavailability of theagent.

Suitable surfactants which can be employed in fabricating the particlesof the invention include but are not limited to hexadecanol; fattyalcohols such as polyethylene glycol (PEG); polyoxyethylene-9-laurylether; a surface active fatty acid, such as palmitic acid or oleic acid;glycocholate; surfactin; a poloxomer; a sorbitan fatty acid ester suchas sorbitan trioleate (Span 85); and tyloxapol.

The surfactant can be present in the particles in an amount ranging fromabout 0 to about 60 weight %. Preferably, it can be present in theparticles in an amount ranging from about 5 to about 50 weight %.

It is understood that when the particles includes a carboxylic acid, amultivalent salt, an amino acid, a surfactant or any combination thereofthat interaction between these components of the particle and thecharged lipid can occur.

The particles, also referred to herein as powder, can be in the form ofa dry powder suitable for inhalation. In a particular embodiment, theparticles can have a tap density of less than about 0.4 g/cm³. Particleswhich have a tap density of less than about 0.4 g/cm³ are referred toherein as “aerodynamically light particles.” More preferred areparticles having a tap density less than about 0.1 g/cm³.

Aerodynamically light particles have a preferred size, e.g., a volumemedian geometric diameter (VMGD) of at least about 5 microns (μm). Inone embodiment, the VMGD is from about 5 μm to about 30 μm. In anotherembodiment of the invention, the particles have a VMGD ranging fromabout 9 μm to about 30 μm. In other embodiments, the particles have amedian diameter, mass median diameter (MMD), a mass median envelopediameter (MMED) or a mass median geometric diameter (MMGD) of at least 5μm, for example from about 5 μm to about 30 μm.

Aerodynamically light particles preferably have “mass median aerodynamicdiameter” (MMAD), also referred to herein as “aerodynamic diameter”,between about 1 μm and about 5 μm. In one embodiment of the invention,the MMAD is between about 1 μm and about 3 μm. In another embodiment,the MMAD is between about 3 μm and about 5 μm.

In another embodiment of the invention, the particles have an envelopemass density, also referred to herein as “mass density” of less thanabout 0.4 g/cm³. The envelope mass density of an isotropic particle isdefined as the mass of the particle divided by the minimum sphereenvelope volume within which it can be enclosed. Tap density can bemeasured by using instruments known to those skilled in the art such asthe Dual Platform Microprocessor Controlled Tap Density Tester (Vankel,N.C.) or a GeoPyc™ instrument (Micrometrics Instrument Corp., Norcross,Ga. 30093). Tap density is a standard measure of the envelope massdensity. Tap density can be determined using the method of USP BulkDensity and Tapped Density, United States Pharmacopia convention,Rockville, Md., 10^(th) Supplement, 4950-4951, 1999. Features which cancontribute to low tap density include irregular surface texture andporous structure.

The diameter of the particles, for example, their VMGD, can be measuredusing an electrical zone sensing instrument such as a Multisizer IIe,(Coulter Electronic, Luton, Beds, England), or a laser diffractioninstrument (for example Helos, manufactured by Sympatec, Princeton,N.J.). Other instruments for measuring particle diameter are well knownin the art. The diameter of particles in a sample will range dependingupon factors such as particle composition and methods of synthesis. Thedistribution of size of particles in a sample can be selected to permitoptimal deposition within targeted sites within the respiratory tract.

Experimentally, aerodynamic diameter can be determined by employing agravitational settling method, whereby the time for an ensemble ofparticles to settle a certain distance is used to infer directly theaerodynamic diameter of the particles. An indirect method for measuringthe mass median aerodynamic diameter (MMAD) is the multi-stage liquidimpinger (MSLI).

The aerodynamic diameter, d_(aer), can be calculated from the equation:

d _(aer) =d _(g)√ρ√_(tap)

where d_(g) is the geometric diameter, for example the MMGD and ρ is thepowder density.

Particles which have a tap density less than about 0.4 g/cm³, mediandiameters of at least about 5 μm, and an aerodynamic diameter of betweenabout 1 μm and about 5 μm, preferably between about 1 μm and about 3 μm,are more capable of escaping inertial and gravitational deposition inthe oropharyngeal region, and are targeted to the airways or the deeplung. The use of larger, more porous particles is advantageous sincethey are able to aerosolize more efficiently than smaller, denseraerosol particles such as those currently used for inhalation therapies.

In comparison to smaller particles the larger aerodynamically lightparticles, preferably having a VMGD of at least about 5 μm, also canpotentially more successfully avoid phagocytic engulfment by alveolarmacrophages and clearance from the lungs, due to size exclusion of theparticles from the phagocytes' cytosolic space. Phagocytosis ofparticles by alveolar macrophages diminishes precipitously as particlediameter increases beyond about 3 Kawaguchi, H., et al., Biomaterials,7: 61-66 (1986); Krenis, L. J. and Strauss, B., Proc. Soc. Exp. Med.,107: 748-750 (1961); and Rudt, S. and Muller, R. H., J. Contr. Rel., 22:263-272 (1992). For particles of statistically isotropic shape, such asspheres with rough surfaces, the particle envelope volume isapproximately equivalent to the volume of cytosolic space requiredwithin a macrophage for complete particle phagocytosis.

The particles may be fabricated with the appropriate material, surfaceroughness, diameter and tap density for localized delivery to selectedregions of the respiratory tract such as the deep lung or upper orcentral airways. For example, higher density or larger particles may beused for upper airway delivery, or a mixture of varying sized particlesin a sample, provided with the same or different therapeutic agent maybe administered to target different regions of the lung in oneadministration. Particles having an aerodynamic diameter ranging fromabout 3 to about 5 μm are preferred for delivery to the central andupper airways. Particles having an aerodynamic diameter ranging fromabout 1 to about 3 μm are preferred for delivery to the deep lung.

Inertial impaction and gravitational settling of aerosols arepredominant deposition mechanisms in the airways and acini of the lungsduring normal breathing conditions. Edwards, D. A., J. Aerosol Sci., 26:293-317 (1995). The importance of both deposition mechanisms increasesin proportion to the mass of aerosols and not to particle (or envelope)volume. Since the site of aerosol deposition in the lungs is determinedby the mass of the aerosol (at least for particles of mean aerodynamicdiameter greater than approximately 1 μm), diminishing the tap densityby increasing particle surface irregularities and particle porositypermits the delivery of larger particle envelope volumes into the lungs,all other physical parameters being equal.

The low tap density particles have a small aerodynamic diameter incomparison to the actual envelope sphere diameter. The aerodynamicdiameter, d_(aer), is related to the envelope sphere diameter, d (Gonda,I., “Physico-chemical principles in aerosol delivery,” in Topics inPharmaceutical Sciences 1991 (eds. D. J. A. Crommelin and K. K. Midha),pp. 95-117, Stuttgart: Medpharm Scientific Publishers, 1992)), by theformula:

d _(aer) =d√ρ

where the envelope mass ρ is in units of g/cm³. Maximal deposition ofmonodispersed aerosol particles in the alveolar region of the human lung(˜60%) occurs for an aerodynamic diameter of approximately d_(aer)=3 μm.Heyder, J. et al., J. Aerosol Sci., 17: 811-825 (1986). Due to theirsmall envelope mass density, the actual diameter d of aerodynamicallylight particles comprising a monodisperse inhaled powder that willexhibit maximum deep-lung deposition is:

d=3√ρμm(where ρ<1 g/cm³);

where d is always greater than 3 μm. For example, aerodynamically lightparticles that display an envelope mass density, ρ=0.1 g/cm³, willexhibit a maximum deposition for particles having envelope diameters aslarge as 9.5 μm. The increased particle size diminishes interparticleadhesion forces. Visser, J., Powder Technology, 58: 1-10. Thus, largeparticle size increases efficiency of aerosolization to the deep lungfor particles of low envelope mass density, in addition to contributingto lower phagocytic losses.

The aerodyanamic diameter can be calculated to provide for maximumdeposition within the lungs, previously achieved by the use of verysmall particles of less than about five microns in diameter, preferablybetween about one and about three microns, which are then subject tophagocytosis. Selection of particles which have a larger diameter, butwhich are sufficiently light (hence the characterization“aerodynamically light”), results in an equivalent delivery to thelungs, but the larger size particles are not phagocytosed. Improveddelivery can be obtained by using particles with a rough or unevensurface relative to those with a smooth surface.

Suitable particles can be fabricated or separated, for example byfiltration or centrifugation, to provide a particle sample with apreselected size distribution. For example, greater than about 30%, 50%,70%, or 80% of the particles in a sample can have a diameter within aselected range of at least about 5 μm. The selected range within which acertain percentage of the particles must fall may be for example,between about 5 and about 30 μm, or optimally between about 5 and about15 μm. In one preferred embodiment, at least a portion of the particleshave a diameter between about 9 and about 11 μm. Optionally, theparticle sample also can be fabricated wherein at least about 90%, oroptionally about 95% or about 99%, have a diameter within the selectedrange. The presence of the higher proportion of the aerodynamicallylight, larger diameter particles in the particle sample enhances thedelivery of therapeutic or diagnostic agents incorporated therein to thedeep lung. Large diameter particles generally mean particles having amedian geometric diameter of at least about 5 μm.

The particles can be prepared by spray drying. For example, a spraydrying mixture, also referred to herein as “feed solution” or “feedmixture”, which includes the bioactive agent and one or more chargedlipids having a charge opposite to that of the active agent uponassociation are fed to a spray dryer.

For example, when employing a protein active agent, the agent may bedissolved in a buffer system above or below the pI of the agent.Specifically, insulin for example may be dissolved in an aqueous buffersystem (e.g., citrate, phosphate, acetate, etc.) or in 0.01 N HCl. ThepH of the resultant solution then can be adjusted to a desired valueusing an appropriate base solution (e.g., 1 N NaOH). In one preferredembodiment, the pH may be adjusted to about pH 7.4. At this pH insulinmolecules have a net negative charge (pI=5.5). In another embodiment,the pH may be adjusted to about pH 4.0. At this pH insulin moleculeshave a net positive charge (pI=5.5). Typically the cationic phospholipidis dissolved in an organic solvent or combination of solvents. The twosolutions are then mixed together and the resulting mixture is spraydried.

For a small molecule active agent, the agent may be dissolved in abuffer system above or below the pKa of the ionizable group(s).Specifically, albuterol sulfate or estrone sulfate, for example, can bedissolved in an aqueous buffer system (e.g., citrate, phosphate,acetate, etc.) or in sterile water for irrigation. The pH of theresultant solution then can be adjusted to a desired value using anappropriate acid or base solution. If the pH is adjusted to about pH 3to about pH 8 range, estrone sulfate will possess one negative chargeper molecule and albuterol sulfate will possess one positive charge permolecule. Therefore, charge interaction can be engineered by the choiceof an appropriate phospholipid. Typically the negatively charged or thepositively charged phospholipid is dissolved in an organic solvent orcombination of solvents and the two solutions are then mixed togetherand the resulting mixture is spray dried.

Suitable organic solvents that can be present in the mixture being spraydried include, but are not limited to, alcohols for example, ethanol,methanol, propanol, isopropanol, butanols, and others. Other organicsolvents include, but are not limited to, perfluorocarbons,dichloromethane, chloroform, ether, ethyl acetate, methyl tert-butylether and others. Aqueous solvents that can be present in the feedmixture include water and buffered solutions. Both organic and aqueoussolvents can be present in the spray-drying mixture fed to the spraydryer. In one embodiment, an ethanol water solvent is preferred with theethanol:water ratio ranging from about 50:50 to about 90:10. The mixturecan have a, acidic or alkaline pH. Optionally, a pH buffer can beincluded. Preferably, the pH can range from about 3 to about 10.

The total amount of solvent or solvents being employed in the mixturebeing spray dried generally is greater than 99 weight percent. Theamount of solids (drug, charged lipid and other ingredients) present inthe mixture being spray dried generally is less than about 1.0 weightpercent. Preferably, the amount of solids in the mixture being spraydried ranges from about 0.05% to about 0.5% by weight.

Using a mixture which includes an organic and an aqueous solvent in thespray drying process allows for the combination of hydrophilic andhydrophobic components, while not requiring the formation of liposomesor other structures or complexes to facilitate solubilization of thecombination of such components within the particles.

Suitable spray-drying techniques are described, for example, by K.Masters in “Spray Drying Handbook”, John Wiley & Sons, New York, 1984.Generally, during spray-drying, heat from a hot gas such as heated airor nitrogen is used to evaporate the solvent from droplets formed byatomizing a continuous liquid feed. Other spray-drying techniques arewell known to those skilled in the art. In a preferred embodiment, arotary atomizer is employed. An example of a suitable spray dryer usingrotary atomization includes the Mobile Minor spray dryer, manufacturedby Niro, Denmark. The hot gas can be, for example, air, nitrogen orargon.

Preferably, the particles of the invention are obtained by spray dryingusing an inlet temperature between about 100° C. and about 400° C. andan outlet temperature between about 50° C. and about 130° C.

The spray dried particles can be fabricated with a rough surface textureto reduce particle agglomeration and improve flowability of the powder.The spray-dried particle can be fabricated with features which enhanceaerosolization via dry powder inhaler devices, and lead to lowerdeposition in the mouth, throat and inhaler device.

The particles of the invention can be employed in compositions suitablefor drug delivery via the pulmonary system. For example, suchcompositions can include the particles and a pharmaceutically acceptablecarrier for administration to a patient, preferably for administrationvia inhalation. The particles can be co-delivered with larger carrierparticles, not including a therapeutic agent, the latter possessing massmedian diameters for example in the range between about 50 μm and about100 μm. The particles can be administered alone or in any appropriatepharmaceutically acceptable carrier, such as a liquid, for examplesaline, or a powder, for administration to the respiratory system.

Particles including a medicament, for example one or more of the drugslisted above, are administered to the respiratory tract of a patient inneed of treatment, prophylaxis or diagnosis. Administration of particlesto the respiratory system can be by means such as known in the art. Forexample, particles are delivered from an inhalation device. In apreferred embodiment, particles are administered via a dry powderinhaler (DPI). Metered-dose-inhalers (MDI), nebulizers or instillationtechniques also can be employed.

Various suitable devices and methods of inhalation which can be used toadminister particles to a patient's respiratory tract are known in theart. For example, suitable inhalers are described in U.S. Pat. No.4,069,819, issued Aug. 5, 1976 to Valentini, et al., U.S. Pat. No.4,995,385 issued Feb. 26, 1991 to Valentini, et al., and U.S. Pat. No.5,997,848 issued Dec. 7, 1999 to Patton, et al. Various suitable devicesand methods of inhalation which can be used to administer particles to apatient's respiratory tract are known in the art. For example, suitableinhalers are described in U.S. Pat. Nos. 4,995,385, and 4,069,819 issuedto Valentini, et al., U.S. Pat. No. 5,997,848 issued to Patton. Otherexamples include, but are not limited to, the Spinhaler® (Fisons,Loughborough, U.K.), Rotahaler® (Glaxo-Wellcome, Research TriangleTechnology Park, North Carolina), FlowCaps® (Hovione, Loures, Portugal),Inhalator® (Boehringer-Ingelheim, Germany), and the Aerolizer®(Novartis, Switzerland), the diskhaler (Glaxo-Wellcome, RTP, NC) andothers, such as known to those skilled in the art. Preferably, theparticles are administered as a dry powder via a dry powder inhaler.

Preferably, particles administered to the respiratory tract travelthrough the upper airways (oropharynx and larynx), the lower airwayswhich include the trachea followed by bifurcations into the bronchi andbronchioli and through the terminal bronchioli which in turn divide intorespiratory bronchioli leading then to the ultimate respiratory zone,the alveoli or the deep lung. In a preferred embodiment of theinvention, most of the mass of particles deposits in the deep lung. Inanother embodiment of the invention, delivery is primarily to thecentral airways. Delivery to the upper airways can also be obtained.

In one embodiment of the invention, delivery to the pulmonary system ofparticles is in a single, breath-actuated step, as described in U.S.patent application, High Efficient Delivery of a Large Therapeutic MassAerosol, application Ser. No. 09/591,307, filed Jun. 9, 2000, which isincorporated herein by reference in its entirety. In another embodimentof the invention, at least 50% of the mass of the particles stored inthe inhaler receptacle is delivered to a subject's respiratory system ina single, breath-activated step. In a further embodiment, at least 5milligrams and preferably at least 10 milligrams of a medicament isdelivered by administering, in a single breath, to a subject'srespiratory tract particles enclosed in the receptacle. Amounts as highas 15, 20, 25, 30, 35, 40 and 50 milligrams can be delivered.

As used herein, the term “effective amount” means the amount needed toachieve the desired therapeutic or diagnostic effect or efficacy. Theactual effective amounts of drug can vary according to the specific drugor combination thereof being utilized, the particular compositionformulated, the mode of administration, and the age, weight, conditionof the patient, and severity of the symptoms or condition being treated.Dosages for a particular patient can be determined by one of ordinaryskill in the art using conventional considerations, (e.g. by means of anappropriate, conventional pharmacological protocol). For example,effective amounts of albuterol sulfate range from about 100 microgramsGO to about 10 milligrams (mg).

Aerosol dosage, formulations and delivery systems also may be selectedfor a particular therapeutic application, as described, for example, inGonda, I. “Aerosols for delivery of therapeutic and diagnostic agents tothe respiratory tract,” in Critical Reviews in Therapeutic Drug CarrierSystems, 6: 273-313, 1990; and in Moren, “Aerosol dosage forms andformulations,” in: Aerosols in Medicine. Principles, Diagnosis andTherapy, Moren, et al., Eds, Esevier, Amsterdam, 1985.

Drug release rates can be described in terms of release constants. Thefirst order release constant can be expressed using the followingequations:

M _((t)) =M _((∞))*(1−e ^(−k*t))  (1)

Where k is the first order release constant. M_((∞)) is the total massof drug in the drug delivery system, e.g. the dry powder, and M_((t)) isthe amount of drug mass released from dry powders at time t.

Equations (1) may be expressed either in amount (i.e., mass) of drugreleased or concentration of drug released in a specified volume ofrelease medium.

For example, Equation (1) may be expressed as:

C _((t)) =C _((∞))*(1−e ^(−k*t)) or Release_((t))=Release_((∞))*(1−e^(−k*t))  (2)

Where k is the first order release constant. C_((∞)) is the maximumtheoretical concentration of drug in the release medium, and C_((t)) isthe concentration of drug being released from dry powders to the releasemedium at time t.

Drug release rates in terms of first order release constant can becalculated using the following equations:

k=−ln(M _((∞)) −M _((t)))/M _((∞)) /t  (3)

The release constants presented in Tables 4 and 8 employ equation (2).

As used herein, the term “a” or “an” refers to one or more.

The term “nominal dose” as used herein, refers to the total mass ofbioactive agent which is present in the mass of particles targeted foradministration and represents the maximum amount of bioactive agentavailable for administration.

EXEMPLIFICATION Materials

Humulin L (human insulin zinc suspension) was obtained from Lilly (100U/mL)

Mass Median Aerodynamic Diameter-MMAD (μm)

The mass median aerodynamic diameter was determined using anAerosizer/Aerodisperser (Amherst Process Instrument, Amherst, Mass.).Approximately 2 mg of powder formulation was introduced into theAerodisperser and the aerodynamic size was determined by time of flightmeasurements.

Volume Median Geometric Diameter-VMGD (μm)

The volume median geometric diameter was measured using a RODOS drypowder disperser (Sympatec, Princeton, N.J.) in conjunction with a HELOSlaser diffractometer (Sympatec). Powder was introduced into the RODOSinlet and aerosolized by shear forces generated by a compressed airstream regulated at 2 bar. The aerosol cloud was subsequently drawn intothe measuring zone of the HELOS, where it scattered light from a laserbeam and produced a fraunhofer diffraction pattern used to infer theparticle size distribution and determine the median value.

Where noted, the volume median geometric diameter was determined using aCoulter Multisizer II. Approximately 5-10 mg powder formulation wasadded to 50 mL isoton II solution until the coincidence of particles wasbetween 5 and 8%.

Determination of Plasma Insulin Levels

Quantification of insulin in rat plasma was performed using a humaninsulin specific RIA kit (Linco Research, Inc., St. Charles, Mo.,catalog #HI-14K). The assay shows less than 0.1% cross reactivity withrat insulin. The assay kit procedure was modified to accommodate the lowplasma volumes obtained from rats, and had a sensitivity ofapproximately 5 μU/mL.

Determination of Estrone-Sulfate Plasma Levels

Quantification of estrone-sulfate in rat plasma was performed using anestrone-sulfate RIA kit (Diagnostic Systems Laboratories, Inc., Webster,Tex., catalog #DSL-05400). The assay kit procedure was modified toaccommodate the low plasma volumes obtained from rats and to correct forinfluence of the human serum standard matrix, and had a sensitivity ofapproximately 0.025 ng/mL.

Preparation of Insulin Formulations

The powder formulations listed in Table 1 were prepared as follows.Pre-spray drying solutions were prepared by dissolving the lipid inethanol and the insulin, leucine, and/or sodium citrate in water. Theethanol solution was then mixed with the water solution at a ratio of60/40 ethanol water. Final total solute concentration of the solutionused for spray drying varied from 1 g/L to 3 g/L. As an example, theDPPC/citrate/insulin (60/10/30) spray drying solution was prepared bydissolving 600 mg DPPC in 600 mL of ethanol, dissolving 100 mg of sodiumcitrate and 300 mg of insulin in 400 mL of water and then mixing the twosolutions to yield one liter of cosolvent with a total soluteconcentration of 1 g/L (w/v). Higher solute concentrations of 3 g/L(w/v) were prepared by dissolving three times more of each solute in thesame volumes of ethanol and water.

The solution was then used to produce dry powders. A Nitro AtomizerPortable Spray Dryer (Niro, Inc., Columbus, Md.) was used. Compressedair with variable pressure (1 to 5 bar) ran a rotary atomizer (2,000 to30,000 rpm) located above the dryer. Liquid feed with varying rate (20to 66 mL/min) was pumped continuously by an electronic metering pump(LMI, Model #A151-192s) to the atomizer. Both the inlet and outlettemperatures were measured. The inlet temperature was controlledmanually; it could be varied between 100° C. and 400° C. and wasestablished at 100, 110, 150, 175 or 200° C., with a limit of control of5° C. The outlet temperature was determined by the inlet temperature andsuch factors as the gas and liquid feed rates (it varied between 50° C.and 130° C.). A container was tightly attached to the cyclone forcollecting the powder product.

TABLE 1 POWDER FORMU- LATION COMPOSITION (%) NUMBER DPePC DSePC DPPGDPPC Leucine Citrate Insulin  1† 70 10 20 2 70 20 10 3 70 10 20 4 50 50  5 ‡ 40 10 50 6 70 10 20 7 50 50 8 54.5 45.5 9 50 10 40 10  70 10 2 11 70 8 2 20  12 † 40 10 50 13† 60 10 30    13A‡ 60 10 30 14‡ 70 20 15† 7020 10 †Lots # 4-xxx-201002 (#1), 4-XXX-201 065 (#12), 04-00024 (#13),4-xxx-114068C (#13A) and 4-xxx-167113 (#15), which contain the lipidDPPC, serve as negative controls. ‡ Powder formulation #5 was spraydried at pH = 4.0. ‡Powder formulation #14 was spray dried at pH = 7.4.

The physical characteristic of the insulin containing powders is setforth in Table 2. The MMAD and VMGD were determined as detailed above.

TABLE 2 COMPOSITIONS MMAD VMGD Density Formulations (% WEIGHT BASIS)(μm) § (μm) ¶ (g/cc) ‡ Humulin R — — — — Humulin L — — — — Humulin U — —— —  1 DPPC/Leu/Insulin 2.6 13.4 0.038 (Sigma) = 70/10/20  2 DSePC(Avanti)/Leu/Insulin 3.3 10.0 0.109 (Sigma) = 70/10/20  3 DSePC(Avanti)/Leu/Insulin 3.4 13.6 0.063 (Sigma) = 70/10/20  4 DPePC(Avanti)/Insulin 3.2 15.3 0.044 (Sigma) = 50/50  5 DPPG/Sodium Citrate/3.9 11.6 0.113 Insulin = 40/10/50  6 DPePC (Genzyme)/Leu/ 2.6 9.1 0.082Insulin (BioBras) =   70/10/20  7 DPePC (Avanti)/Insulin 2.8 11.4 0.060(BioBras) = 50/50  8 DPePC (Genzyme)/Insulin 2.8 12.6 0.049 (BioBras) =54.5/45.5  9 DPePC (Genzyme)/Leu/ 2.2 8.4 0.069 Insulin (BioBras) =50/10/40 10 DPePC (Avanti)/Leu/Insulin 3.7 15.5 0.057 (BioBras) =70/10/20 11 DPePC (Avanti)/Leu/Sodium 2.6 15.3 0.029 Citrate/Insulin(BioBras) = 70/8/2/20 12 DPPC/Sodium Citrate/ 3.5 11.6 0.091 Insulin =40/10/50 13 DPPC/Insulin/Sodium 1.9 8.0 0.056 Citrate = 60/30/10 † Usedas a control formulation for comparison in either in vitro or in vivostudies. § Mass median aerodynamic diameter ¶ Volumetric mediangeometric diameter at 2 bar pressure ‡ Determinedusing d_(aer) = dg√ρ

The data presented in Table 2 showing the physical characteristics ofthe formulations comprising insulin are predictive of the respirabilityof the formulations. That is, as discussed above the large geometricdiameters, small aerodynamic diameters and low densities possessed bythe powder prepared as described herein render the particles respirable.

In Vivo Insulin Experiments

The following experiment was performed to determine the rate and extentof insulin absorption into the blood stream of rats following pulmonaryadministration of dry powder formulations comprising insulin to rats.

The nominal insulin dose administered was 100 μg per rat. To achieve thenominal doses, the total weight of powder administered per rat rangedfrom 0.2 mg to 1 mg, depending on percent composition of each powder.Male Sprague-Dawley rats were obtained from Taconic Farms (Germantown,N.Y.). At the time of use, the animals weighed 386 g in average (+5 gS.E.M.). The animals were allowed free access to food and water.

The powders were delivered to the lungs using an insufflator device forrats (PennCentury, Philadelphia, Pa.). The powder amount was transferredinto the insufflator sample chamber. The delivery tube of theinsufflator was then inserted through the mouth into the trachea andadvanced until the tip of the tube was about a centimeter from thecarina (first bifurcation). The volume of air used to deliver the powderfrom the insufflator sample chamber was 3 mL, delivered from a 10 mLsyringe. In order to maximize powder delivery to the rat, the syringewas recharged and discharged two more times for a total of three airdischarges per powder dose.

The injectable insulin formulation Humulin L was administered viasubcutaneous injection, with an injection volume of 7.2 μL for a nominaldose of 25 μg insulin. Catheters were placed into the jugular veins ofthe rats the day prior to dosing. At sampling times, blood samples weredrawn from the jugular vein catheters and immediately transferred toEDTA coated tubes. Sampling times were 0, 0.25, 0.5, 1, 2, 4, 6, 8, and24 hrs. after powder administration. In some cases an additionalsampling time (12 hrs.) was included, and/or the 24 hr. time pointomitted. After centrifugation, plasma was collected from the bloodsamples. Plasma samples were stored at 4° C. if analysis was performedwithin 24 hours or at −75° C. if analysis would occur later than 24hours after collection. The plasma insulin concentration was determinedas described above.

Table 3 contains the insulin plasma levels quantified using the assaydescribed above.

TABLE 3 PLASMA INSULIN CONCENTRATION (μU/mL) ± S.E.M. Time Humlin (hrs)1 2 3 4 5 6 13A 14 L 15 0   5.0 ±  5.2 ±  5.0 ±  5.0 ±  5.3 ±  5.7 ±  5.0 ±  5.0 ±  5.0 ±  ±5.0 ±  0.0  0.2  0.0  0.0  0.2  0.7  0.0  0.0 0.0  0.0 0.25 1256.4 ± 61.6 ±  98.5 ± 518.2 ± 240.8 ± 206.8 ± 1097.7 ±933.9 ± 269.1 ± 1101.9 ± 144.3  22.5 25.3 179.2 67.6 35.1 247.5 259.782.8 258.9 0.5 1335.8 ± 85.2 ± 136.7 ± 516.8 ± 326.2 ± 177.3 ±  893.5 ±544.9 ± 459.9 ± 1005.4 ± 81.9 21.7 37.6 190.9 166.9   7.8 177.0 221.191.6 263.9 1  859.0 ± 85.4 ± 173.0 ± 497.0 ± 157.3 ± 170.5 ±  582.5 ±229.6 ± 764.7 ±  387.5 ± 199.4  17.6 28.8  93.9 52.5 32.9 286.3  74.4178.8  143.9 2  648.6 ± 94.8 ± 158.3 ± 496.5 ± 167.7 ± 182.2 ±  208.5 ±129.8 ± 204.4 ±  343.8 ± 171.1  25.0 39.1 104.9 70.5 75.0  78.3  45.736.7  95.3 4  277.6 ± 52.5 ±  98.0 ± 343.8 ± 144.8 ± 170.2 ±  34.9 ± 41.9 ±  32.1 ±  170.6 ± 86.8  9.1 24.3  66.7 43.8 56.3  5.4  28.7 22.6 79.9 6  104.0 ± 33.0 ±  58.7 ± 251.2 ±  95.7 ± 159.5 ±  12.3 ±  9.0 ± 11.1 ±  15.4 ± 43.1 10.7  4.1  68.4 27.3 43.4  2.4  2.9  7.5  4.5 8 54.4 ± 30.2 ±  42.5 ±  63.2 ±  52.5 ±  94.8 ±   5.2 ±  5.0 ±  5.5 ±  6.5 ± 34.7  8.1 17.8  16.5 13.7 23.5  0.1  0.0  2.1  0.6 12  17.2 ± 6.5 24  5.0 ±  5.5 ±  0.0  0.3 n 5  5  6   6  6  6  8 

The in vivo release data of Table 3 show that powder formulationscomprising insulin and positively charged lipids (DPePC and DSePC) havesignificantly lower initial burst of insulin than that seen with powderformulations comprising insulin and the lipid DPPC (Formulations 1 and13) and sustained elevated levels at 6 to 8 hours. FIG. 1 sets forth therelease profile for insulin from Formulations 2, 3, 6 and 15.

In addition, the use of charged lipids having a charge which is the sameof the active at neutral pH, can also be employed provided that thepreparation of the spray dried formulation is conducted at a pH wherethe lipid and active agent possess overall charges which are oppositeand are therefore capable of charge interaction. See, for example,Formulations 5 which employs the negatively charged lipid DPPG.Formulation 5 was prepared and spray dried at a pH of about 4.0. At thispH, DPPG is negatively charged and insulin becomes positively charged(pI=5.5) thereby providing for a charge interaction to occur. However,when the DPPG and insulin are prepared and spray dried at pH=7.4 whereboth the DPPG and insulin possess an overall negative charge,Formulation 14, the proper environment for charge interaction to occuris not provided. It is noted that Formulation 5 showed a significantlylower initial burst of insulin (240.8±67.6 μU/mL) as compared toFormulation 14 (933.9±259.7 μU/mL) with higher sustained levels at 6 to8 hours post treatment. FIG. 2 shows a comparison of the in vivo releaseprofile for Formulations 5, 14 and 13A (lipid, DPPC).

In Vitro Analysis of Insulin-Containing Formulations

The in vitro release of insulin containing dry powder formulations wasperformed as described by Gietz et al. in Eur. J. Pharm. Biopharm.,45:259-264 (1998), with several modifications. Briefly, in 20 mLscrew-capped glass scintillation vials about 10 mg of each dry powderformulation was mixed with 4 mL of warm (37° C.) 1% agarose solutionusing polystyrene stir bars. The resulting mixture was then distributedin 1 mL aliquots to a set of five fresh 20 mL glass scintillation vials.The dispersion of dry powder in agarose was cooled in an ambienttemperature dessicator box protected from light to allow gelling.Release studies were conducted on an orbital shaker at about 37° C. Atpredetermined time points, previous release medium (1.5 mL) was removedand fresh release medium (1.5 mL) was added to each vial. Typical timepoints for these studies were 5 minutes, 1, 2, 4, 6 and 24 hours. Therelease medium used consisted of 20 mM4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES), 138 mMNaCl, 0.5% Pluronic (Synperonic PE/F68; to prevent insulin filbrillationin the release medium); pH 7.4. A Pierce (Rockford, Ill.) protein assaykit (See Anal Biochem, 150:76-85 (1985)) using known concentrations ofinsulin standard was used to monitor insulin concentrations in therelease medium.

Table 4 summarizes the in vitro release data and first order releaseconstants for powder formulations of Table 1 comprising insulin.

TABLE 4 Cumulative Cumulative Maximum ‡ First Order ‡ Powder % Insulin %Insulin Release Release Formulation Released Released at 24 hr ConstantsNumber at 6 hr at 24 hr (Cumulative %) (hr⁻¹) Humulin R 92.67 ± 0.3694.88 ± 0.22  91.6 ± 5.42 1.0105 ± 0.2602 Humulin L 19.43 ± 0.41 29.71 ±0.28  36.7 ± 2.56 0.0924 ± 0.0183 Humulin U  5.17 ± 0.18 12.65 ± 0.43 46.6 ± 27.0 0.0158 ± 0.0127  2 31.50 ± 0.33 47.52 ± 0.43 48.22 ± 0.460.1749 ± 0.0038  3 26.34 ± 0.71 37.49 0.27 38.08 ± 0.72 0.1837 ± 0.0079 4 24.66 ± 0.20 31.58 ± 0.33 31.51 ± 1.14 0.2457 ± 0.0214  5 29.75 ±0.17 35.28 ± 0.19 33.66 ± 2.48 0.4130 ± 0.0878  6 17.04 ± 0.71 24.71 ±0.81 25.19 ± 0.52 0.1767 ± 0.0083  7 13.53 ± 0.19 19.12 ± 0.40 19.51 ±0.48 0.1788 ± 0.0101  8 13.97 ± 0.27 17.81 ± 0.46 17.84 ± 0.55 0.2419 ±0.0178  9 17.47 ± 0.38 22.17 ± 0.22 21.97 ± 0.64 0.2734 ± 0.0196 1025.96 ± 0.31 34.94 ± 0.31 35.43 ± 0.90 0.2051 ± 0.0120 11 34.33 ± 0.5147.21 ± 0.47 47.81 ± 0.85 0.1994 ± 0.0082 12 61.78 ± 0.33 68.56 ± 0.2365.20 ± 3.34 0.5759 ± 0.0988 13 78.47 ± 0.40 85.75 ± 0.63  84.9 ± 3.810.5232 ± 0.0861 ‡ Release _((t)) = Release _((inf)) *(1 − e^(−k)*^(t)) †Used as a control formulation.

The data presented in Table 4 show that for insulin containing powderformulations employing the positively charged lipid DPePC (Formulations4 and 6-11) and DSePC (Formulations 2 and 3), first order releaseconstants similar to that observed with the slow release injectableinsulin formulation, Humulin L, can be achieved. Further, the firstorder release constants of these same formulations is significantlylower than that observed with the fast release injectable insulinformulation, Humulin R. As such, sustained release dry powder insulinformulations having varying compositions of positively charged lipid canbe formulated.

Preparation of Estrone Sulfate-Containing Powder Formulations

The estrone sulfate powder formulations listed in Table were prepared asfollows. Pre-spray drying solutions were prepared by dissolving thelipin in ethanol and estrone sulfate and leucine in water. The ethanolsolution was then mixed with the water solution at a ration 70/30ethanol/water. Final total solute concentration of the solution used forspray drying varied from 1 g/L to 3 g/L. As an example, theDPePC/leucine/estrone sulfate (76/20/4) spray drying solution wasprepared by dissolving 760 mg of DPePC in 700 mL of ethanol, dissolving200 mg of leucine and 40 mg of estrone sulfate in 300 mL of water andthen mixing the two solutions to yield one liter of cosolvent with atotal solute concentration of 1 g/L (w/v). Higher solute concentrationsof, for example, 3 g/L (w/) were prepared by dissolving three times moreof each solute in the same volumes of ethanol and water.

The mixture was spray dried following the procedure described above forthe insulin containing powder formulation. During spray drying, the feedrate was about 50 mL/min, the inlet temperature ranged from about 110°C. to about 120° C., and the outlet temperature was about 52° C.

The physical characteristic of the estrone sulfate containing powders isset forth in Table 5. The MMAD and VMGD were determined as detailedabove.

TABLE 5 POWDER DEN- FORMULATION COMPOSITIONS MMAD VMGD SITY NUMBER (%WEIGHT BASIS) (μm) § (μm) ¶ (g/cc) ‡ 16 DPePC (Avanti)/Leucine/ 5.9 16.00.136 Estrone Sulfate (sodium salt) = 76/20/4 17 DPPC/Leucine/ 3.7 12.7# 0.085 Estrone Sulfate (sodium salt) = 76/20/4 † Used as a control forcomparison for in vivo studies § Mass median aerodynamic diameter ¶Volumetric median geometric diameter at 2 bar pressure # Measured usingCoulter Multisizer ‡ Determined using d_(aer) = d_(g)√ρ

The data presented in Table 5 showing the physical characteristics ofthe formulations comprising estrone sulfate are predictive of therespirability of the formulations. That is, as discussed above the largegeometric diameters, small aerodynamic diameters and low densitiespossessed by the powder prepared as described herein render theparticles respirable.

In Vivo Experiments-Estrone Sulfate Containing Powders

The following experiment was performed to determine the rate and extentof estrone sulfate absorption into the blood stream of rats followingpulmonary administration of dry powder formulations comprising estronesulfate.

The nominal estrone-sulfate dose administered was 40 μg per rat, in 1 mgof powder. Male Sprague-Dawley rats were obtained from Taconic Farms(Germantown, N.Y.). At the time of use, the animals weighed an averageof 415 g (±10 g S.E.M.). The animals were allowed free access to foodand water.

The powders were delivered to the lungs using an insufflator device forrats (PennCentury, Philadelphia, Pa.). The powder amount was transferredinto the insufflator sample chamber. The delivery tube of theinsufflator was then inserted through the mouth into the trachea andadvanced until the tip of the tube was about a centimeter from thecarina (first bifurcation). The volume of air used to deliver the powderfrom the insufflator sample chamber was 3 mL, delivered from a 10 mLsyringe. In order to maximize powder delivery to the rat, the syringewas recharged and discharged two more times for a total of three airdischarges per powder dose.

Catheters were placed into the jugular veins of the rats the day priorto dosing. At sampling times, blood samples were drawn from the jugularvein catheters and immediately transferred to EDTA coated tubes.Sampling times were 0, 0.25, 0.5, 1, 2, 4, and 6 hours after powderadministration. After centrifugation, plasma was collected from theblood samples. Plasma samples were stored at 4° C. if analysis wasperformed within 24 hours or at −75° C. if analysis would occur laterthan 24 hours after collection.

Table 6 contains the estrone sulfate plasma levels quantified using theassay described above.

TABLE 6 PLASMA ESTRONE-SULFATE CONCENTRATION (ng/mL) ± S.E.M. TIME (HRS)FORMULATION 16 FORMULATION 17 0  0.07 ± 0.02  0.08 ± 0.05 0.25 12.07 ±1.96 22.26 ± 8.96 0.5 18.88 ± 2.21 23.39 ± 12.72 1 12.20 ± 3.31 10.59 ±0.61 2  4.65 ± 0.77  3.45 ± 0.63 4  4.02 ± 1.42  0.86 ± 0.10 6  1.49 ±0.48  0.33 ± 0.12 n  4  3

The results presented in Table 6 and depicted graphically in FIG. 3,show that the formulation comprising DPePC (overall positive charge) andestrone sulfate (negative charge) exhibited sustained release of estronesulfate when compared to the formulation employing the lipid DPPC (nooverall net charge) and estrone sulfate. Specifically, at six hours postadministration, the plasma level of estrone sulfate for the DPePCcontaining formulation was 1.49±0.48 ng/mL as compared to 0.33±0.12ng/mL for the DPPC containing formulation.

Preparation of Albuterol-Containing Powder Formulations

The albuterol sulfate powder formulations listed in Table 7, wereprepared as follows. Pre-spray drying solutions were prepared bydissolving the lipid in ethanol and albuterol sulfate and leucine inwater. The ethanol solution was then mixed with the water solution at aratio of 70/30 ethanol/water. Final total solute concentration of thesolution used for spray drying varied from 1 g/L to 3 g/L. As anexample, the DPPC/leucine/albuterol sulfate (76/16/8) spray dryingsolution was prepared by dissolving 760 mg of DPPC in 700 mL of ethanol,dissolving 160 mg leucine and 870 mg of albuterol sulfate in 300 mLwater and then mixing the two solutions to yield one liter of cosolventwith a total solute concentration of 1 g/L (w/v). Higher soluteconcentrations of 3 g/L (w/v) were prepared by dissolving three timesmore of each solute in the same volumes of ethanol and water. Thesolution was spray-dried as described above for the insulin containingformulation. Specifically, the inlet temperature was from about 110° C.to about 140° C., and the outlet temperature ranged from about 45-57° C.

The physical characteristics of the albuterol sulfate containing powdersis set forth in Table 7. The MMAD and VMGD were determined as detailedabove.

TABLE 7 POWDER DEN- FORMULATION COMPOSITIONS MMAD VMGD SITY NUMBER (%WEIGHT BASIS) (μm) § (μm) ¶ (g/cc) ‡ 18 DSPC/Leucine/ 3.3 6.1 0.293Albuterol Sulfate = 76/16/8 19 DSPG/Leucine/ 4.1 6.4 0.410 AlbuterolSulfate = 76/16/8 20 DPPC/Leucine/ 2.8 12.0 0.054 Albuterol Sulfate =76/23/1 21 DPPG/Leucine/ 3.3 7.1 0.216 Albuterol Sulfate = 76/16/8 †Used as a control for comparison in either in vitro or in vivo studies §Mass median aerodynamic diameter ¶ Volumetric median geometric diameterat 2 bar pressure ‡ Determined using d_(aer) = d_(g)√ρ

The data presented in Table 7 showing the physical characteristics ofthe formulations comprising albuterol sulfate are predictive of therespirability of the formulations. That is, as discussed above the largegeometric diameters, small aerodynamic diameters and low densitiespossessed by the formulations prepared as described herein render theformulations respirable.

In Vivo Testing of Albuterol Sulfate Formulations

A whole-body plethysmography method for evaluating pulmonary function inguinea pigs was used to assess the sustained effects of the albuterolsulfate formulations listed in Table 7.

The system used was the BUXCO whole-body unrestrained plethysmographsystem with BUXCO XA pulmonary function software (BUXCO Electronics,Inc., Sharon, Conn.). The method was conducted as described by SilbaughS. A. and Mauderly, J. L., in American Physiological Society, Vol.84:1666-1669 (1984) and Chang, B. T., et at in Journal ofPharmacological and Toxicological Methods, Vol. 39(3):163-168 (1998).This method allows individual animals to be challenged repeatedly overtime with methacholine given by nebulization. A calculated measurementof airway resistance based on flow parameters, the enhanced pause PenHwas used as a marker for protection from methacholine-inducedbronchoconstriction. Baseline pulmonary function (airwayhyperresponsiveness) values were measured prior to any experimentaltreatment. Airway hyperresponsiveness was then assessed in response tosaline and methacholine at various timepoints (2-3, 16 and 24 hours)following administration of albuterol-sulfate formulations. Average PenHis calculated from data collected between 4 and 9 minutes followingchallenge with saline or methacholine. The percent of baseline PenH ateach timepoint is calculated for each experimental animal. Values fromanimals that received the same albuterol sulfate formulation weresubsequently averaged to determine the mean group response (±standarderror) at each timepoint.

The nominal dose of albuterol-sulfate administered was 50 μg for theDPPG-based formulation (#21) and 25 μg for the DPPC-based formulation(#20). To achieve those nominal doses, the total weights of powderadministered were 0.625 mg and 2.5 mg, respectively.

Male Hartley guinea pigs were obtained from Elm Hill Breeding Labs(Chelmsford, Mass.). At the time of use, the animals weighed an averageof 363 g (+5 g S.E.M.). The animals were allowed free access to food andwater. The powder amount was transferred into the insufflator samplechamber (insufflation device for guinea pigs, Penn Century,Philadelphia, Pa.). The delivery tube of the insufflator was insertedthrough the mouth into the trachea and advanced until the tip of thetube was about a centimeter from the carina (first bifurcation). Thevolume of the air used to deliver the powder from the insufflator samplechamber was 3 mL, delivered from a 10 mL syringe. In order to maximizepowder delivery to the guinea pig, the syringe was recharged anddischarged two more times for a total of three air discharges per powderdose. Methacholine challenges were performed at time points 2-3, 16 and24 hours after administration.

FIG. 4 shows that the formulation comprising DPPG (overal negativecharge) and albuterol sulfate (overall positive charge) providedsustained protection against methacholine-induced bronchoconstrictionwhen compared to the formulation comprising DPPC (no overall net charge)and albuterol sulfate for at least 24 hours following administration.

In another experiment, as much as 200 μg of albuterol sulfate in aDPPC-based formulation did not provide prolonged protection againstinduced bronchoconstriction.

In Vitro Release Studies-Albuterol Sulfate

Controlled Release Studies of Albuterol Sulfate were conducted using theCOSTAR™ Brand Transwell Inserts, With Plates, Sterile. The plates wereequipped with 6 wells having an area of 4.7 cm². The insert size was 24mm, the pore size was 3.0 μm. A predetermined amount of the powder to betested (approximately 10-15 mg) was placed into a HPMC Size #2 capsule.The capsule was then placed inside an inhaler and the powder was sprayedon the Transwell insert using an in-house vacuum system. Formulationswere run in triplicate.

After spraying, the insert was placed inside the Transwell platecontaining a volume of 1.8 mL of Phosphate Buffered Saline (pH=7.4)which had previously been equilibrated at 37 C for 30 minutes. TheTranswell plate was hermetically sealed in order to prevent evaporationof the buffer during the experiment.

The Transwell Experiment was carried out in an incubator at 37° C. on anorbital shaker at a speed of 100 min⁻¹. At specified time-pointsthroughout the experiment, 1.8 mL of phosphate buffered saline wasremoved from the Transwell plate. The inserts were then placed into anew Transwell plate containing 1.8 mL of fresh phosphate bufferedsaline. Typical Transwell experiments are conducted for 4 hours. Samplesare withdrawn after 5 min., 15, min., 30, min., 1 h, 1.5 h, 2 h, 3 h,and 4 h.

The amount of albuterol sulfate in the PBS buffer sampled atpredetermined in vitro release time points was quantitated using aRP-HPLC method with Phenomenex Luna 5μ, C8(2), 250×4.6 mm column(Torrance, Calif.) and UV detection at 275 nm.

Table 8 summarizes the in vitro release data and first order releaseconstants for the powder formulations of Table 7 comprising albuterolsulfate. The first order release constants for the powder formulationcomprising DSPG (negatively charged) and albuterol sulfate is about 4time slower compared to the powder formulation comprising DSPC (no netoverall charge) and albuterol sulfate (positive).

TABLE 8 Cumu- Maximum First Powder lative % Release Order Formu- Insulinat 4 hr Release lation Compositions Released (Cumulative ConstantsNumber (% weight basis) at 4 hr %)‡ (hr⁻¹)‡ 18 DSPC/Leucine/Albuterol106.21 ± 105.64 ± 0.20 29.7360 ± Sulfate (sodium salt) =  1.73  0.750476/16/8 19 DSPG/Leucine/Albuterol  97.44 ±  95.13 ± 1.39  7.9334 ±Sulfate (sodium  0.68  0.6877 salt) = 76/16/8 ‡Release _((t)) = Release_((inf)) *(1 − e^(−k)*^(t)) †Used as a control formulation.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. Biocompatible particles for delivery of atherapeutic, prophylactic or diagnostic agent to the pulmonary system;wherein the particles have a tap density of less than 0.4 g/cm³, theparticle size is about 5-10 μm, and the particles have a meanaerodynamic diameter between about 1 μm and about 5 μm.